We are pursuing an HIV vaccine approach based on replication-competent Adenovirus (Ad)-recombinants. The rationale is based on the fact that live attenuated vaccines historically have been the most protective, eliciting essentially life-long immunity. Examples include vaccines for small pox, polio, measles, and yellow fever. We conduct pre-clinical vaccine studies in rhesus macaques and challenge with SIV or SHIV (a chimeric SIV virus containing an HIV envelope), systems that model HIV-infection of humans. We use a prime-boost strategy, first immunizing with a replicating adenovirus (Ad) vector carrying an HIV/SIV gene(s) followed by a boosting with HIV/SIV envelope protein. Ad replicates in epithelial cells that line mucosal inductive sites, and therefore elicits strong, persistent cellular immunity at mucosal effector sites as well as in the blood. We are currently conducting a pre-clinical vaccine study in macaques and are investigating cellular immune responses elicited by the vaccine regimen. Of particular interest is the question of whether female and male macaques developed different cellular immune responses to the vaccine components, as female macaques exhibited better protective efficacy compared to males. While sex differences have been seen with other vaccines, this is the first description of such a difference with a candidate AIDS vaccine. We have also collaborated on a study investigating innate and adaptive cellular responses to the replication-competent Ad vector, and have seen that innate immunity is somewhat suppressed while CD4-positive T cells are transiently activated following immunization. Because non-neutralizing antibody responses have been shown to be contributors to protective efficacy, we are studying effector cells which mediate some of these responses. We continue to explore CD4-dependent natural killer (NK) cells, previously shown to play a role in controlling SIV infection. Currently we are examining whether such responses can be boosted by envelope immunizations during the course of anti-retroviral therapy, and also whether vaccination elicits such responses. NK cells themselves are key effector cells in antibody-mediated cellular cytotoxicity, an activity that has been asociated with vaccine-elicited protective efficacy. Current studies include determining if NK cells in the rhesus macaque model contribute to mucosal immune protection and whether they exhibit memory. Gamma-delta T cells, effector cells that are especially important with regard to mucosal immune protection, are also under study. Finally T follicular helper cells (Tfh cells) are critical for promoting B cell development and maturation and induction of potent antibody responses. We are examining the effects of vaccination on development of such cells which should lead to memory B cells and more persistent antibody responses.